Converting cytoskeletal forces into biochemical signals

项目总结 细胞在其局部环境中感知机械提示，这些提示必须转化为细胞内提示 调节细胞生理和控制基因表达的生化信号。有越来越多的 认识机械信号转导在发育中的关键作用及其 疾病状态下的功能障碍，如癌症。然而，与规范的信号转导相反，细胞力 人们对感知知之甚少，这阻碍了定义机械上不同的机械转导的努力。 途径，描绘其特定的生物学功能，并以治疗为目标。 肌动蛋白细胞骨架，一个由动态肌动蛋白细丝、肌球蛋白马达蛋白和数百种 相关因素，使细胞能够机械地与周围环境接触。细胞骨架是经典的 被理解为作为力产生和传递装置，间接地促进机械- 通过其与介导力信号转换的膜锚定部位的物理连接进行的转导 (例如，细胞-细胞和细胞-基质粘连)。然而，我们和其他人最近报道了可溶性的直接结合 含有LIM(LIN-11，ISL-1和Mec-3)结构域到张力肌动蛋白细丝的串联阵列的胞浆蛋白， 这表明细胞骨架本身可能具有将力量转化为生化信号的能力。这里 我建议检验这样一种假设，即不同的LIM蛋白强制激活的肌动蛋白结合是在 功能上离散的下游机械转导通路。通过细胞分析和生物物理 重组后，我们将研究具有代表性的肌动蛋白如何与LIM蛋白结合(目标1) 和FHL1/2(四个半LIM结构域1/2，Aim 2)在细胞质中介导不同的下游功能 细胞骨架损伤修复和核基因表达调控。然后，我们将创新地 将这些方法与冷冻电子显微镜(Cryo-EM)相结合，以可视化通过以下方式激活的肌动蛋白结合 LIM蛋白质的结构细节(目标3)。我们的研究将建立一种守恒力机制 通过LIM结构域的转导与不同的下游信号结果有关，这可能揭示 细胞骨架机械信号网络模块化组织的一般原则。在 从长远来看，这项工作将使精确剖析LIM蛋白的特定背景生物学功能成为可能 活体，便于严格评估其作为治疗靶点的潜力。

PROJECT SUMMARY Cells perceive mechanical cues in their local environments, which must be converted into intracellular biochemical signals to modulate cellular physiology and control gene expression. There is increasing appreciation for mechanical signal transduction’s (“mechanotransduction”) critical role in development and its dysfunction in disease states such as cancer. However, in contrast to canonical signal transduction, cellular force sensing is poorly understood, hampering efforts to define mechanistically distinct mechanotransduction pathways, delineate their specific biological functions, and target them therapeutically. The actin cytoskeleton, a network of dynamic actin filaments, myosin motor proteins, and hundreds of associated factors, enables cells to mechanically interface with their surroundings. The cytoskeleton is classically understood to serve as a force generation and transmission apparatus that indirectly facilitates mechano- transduction through its physical linkages to membrane-anchored sites which mediate force signal conversion (e.g. cell-cell and cell-matrix adhesions). However, we and others have recently reported direct binding of soluble cytosolic proteins containing tandem arrays of LIM (LIN-11, Isl-1 & Mec-3) domains to tensed actin filaments, suggesting that the cytoskeleton itself may have the capacity to transduce forces into biochemical signals. Here I propose to test the hypothesis that force-activated actin binding by distinct LIM proteins is upstream of functionally discrete downstream mechanotransduction pathways. Through cellular assays and biophysical reconstitution, we will investigate how the representative force-activated actin binding LIM proteins zyxin (Aim 1) and FHL1/2 (Four-and-a-Half LIM domains 1/2, Aim 2) mediate distinct downstream functions in cytoplasmic cytoskeletal damage repair and nuclear gene expression regulation, respectively. We will then innovatively interface these approaches with cryo-electron microscopy (cryo-EM) to visualize force-activated actin binding by LIM proteins in structural detail (Aim 3). Our studies will establish how a conserved mechanism of force transduction through LIM domains is linked to distinct downstream signaling outcomes, which is likely to reveal general principles underlying the modular organization of cytoskeletal mechanical signaling networks. In the longer term, this work will enable precision dissection of context-specific biological functions of LIM proteins in vivo, facilitating rigorous evaluation of their potential as therapeutic targets.